A dipeptidyl aminopeptidase method for polypeptide sequencing using gas chromatography-mass spectroscopy for dipeptide identification has been further developed. A new dipeptidyl aminopeptidase has been discovered which cleaves amide bonds involving the imino group of proline. When used in conjunction with the previously reported DAP-I and IV, it should be possible for the first time to confidently degrade any polypeptide. The use of the combined DAP enzymes and our GC-MS method for dipeptide analysis appears to be a superior sequencing technique with sensitivity and speed as the major assets.